Roscigno, Giuseppina (2014) miR-221 acts as stemness promoter in breast cancer cells by targeting DNMT3b. [Tesi di dottorato]

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Abstract

Current hypotheses suggest that tumor originates from cells that carry out a process of “malignant reprogramming” driven by genetic and epigenetic alterations. Multiples studies reported the existence of stem-cell-like cells that acquire the ability to self-renew and are able to generate the bulk of more differentiated cells that form the tumor. This population of cancer cells, called cancer stem cells (CSC), is responsible for sustaining the tumor growth and is able to disseminate and migrate giving metastases to distant organs. Furthermore, CSCs have shown to be more resistant to anti-tumor treatments than the differentiated cells, suggesting that surviving CSCs could be responsible for tumor relapse after therapy. Nevertheless, the cancer stem-like properties are not well characterized yet. MicroRNAs (miRs) are small, noncoding RNAs (20-25 nucleotidies) that play a crucial role in biological processes including development, proliferation, and apoptosis. Previous investigations have linked miRs to the control of self-renewal and differentiation of normal stem cells. The aim of this study was to test the functional role of miRs in human Breast Cancer Stem Cells (BCSCs) also named mammospheres. We analyzed, by miR-Array, the miRs differentially expressed in BCSCs and their differentiated counterpart. Among several miRs, we focused our attention on miR-221 that was found increased in mammospheres. In order to validate data achieved in primary cultures, we obtained mammospheres from T47D, an immortalized breast cancer cell line. Interestingly, like primary cultures mammospheres, also T47D mammospheres exhibited increased levels of miR-221 compared to T47D differentiated cells. Moreover, the overexpression of miR-221 by a miR mimic in T47D differentiated cells was able to increase the number of mammospheres and the expression of stem cell protein markers. Among miR-221 targets, we demonstrated, by luciferase-assay, that miR-221 targets the 3' untranslated region of DNMT3b, a DNA Methyl Transferase. Furthermore, our data showed that DNMT3b was able to repress the expression of some stemness genes, such as Nanog and Oct3/4, and mammospheres formation partially reverting miR-221 mediated effects on stemness properties. In conclusion, we hypothesize that miR-221 may contribute to breast cancer tumorigenicity regulating the stemness properties through DNMT3b expression.

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